Abstract: The developmental mechanisms of human and Drosophila blood systems show remarkable parallels. Most genes essential for the formation and maturation of Drosophila hemocytes are conserved in humans, and the majority is associated with hematopoietic malignancies. We have identified a new gene, zfrp8, controlling cell proliferation of the hematopoietic organs, the lymph glands. Several loss-of-function alleles in zfrp8 cause enormous hyperplasia of the lymph glands, over-proliferation of immature blood cells, and severe growth delay in other tissues. Zfrp8 and its human ortholog, PDCD2, are >50% homologous. PDCD2 appears to be involved in human blood malignancies. This conclusion is based on its regulation by the BCL6 (B-cell lymphoma 6) oncogene and its chromosomal localization to a region of chromosome 6q27, frequently deleted in human lymphomas. In some lymphomas, PDCD2 is present in the cytoplasm of normal lymphocytes, but is not detected in malignant lymphocytes, indicating that the gene may have similar function in cell proliferation in Drosophila and humans. In the bone marrow and bloth delay in other tissues. Zfrp8 and its human ortholog, PDCD2, are >50% homologous. PDCD2 appears to be involved in human blood malignancies. This conclusion is based on its regulation by the BCL6 (B-cell lymphoma 6) oncogene and its chromosomal localization to a region of chromosome 6q27, frequently deleted in human lymphomas. In some lymphomas, PDCD2 is present in the cytoplasm of normal lymphocytes, but is not detected in malignant lymphocytes, indicating that the gene may have similar function in cell proliferation in Drosophila and humans. In the bone marrow and blood of leukemia patients, we have detected high levels of a smaller form of PDCD2, PDCD2[unreadable]33, barely detected in samples from healthy controls. High levell cycle control. We have found that pannier, encoding a GATA factor, has the opposite lymph gland phenotype of zfrp8 and that the two genes interact genetically. The PDCD2 protein is highly conserved in humans and Drosophila. PDCD2 was originally identified in mammalian cells as an apoptosis-associated gene, but our results suggest that also in vertebrates it is associated with cell proliferation. PDCD2 is negatively regulated by the BCL6 oncogene that is localized to a region of chromosome 6q frequently deleted in human lymphomas. Malignant lymphocytes show high levels of BCL6 expression, but PDCD2 is not detected, although it is found in the cytoplasm of normal lymphocytes. In normal bone marrow we find a 48kD form of PDCD2, but in bone marrow and blood of leukemia patients, we find high levels of a smaller form of the protein (44kD), barely detected in blood from healthy controls. This smaller form of PDCD2 is strongly present in all cancer cell lines we have tested and correlates with increased levels of cell proliferation and oncogenesis. These results suggest that Zfrp8/PDCD2, in both flies and humans, has the same mechanistic function in cell proliferation and blood development. Our proposed experiments will elucidate how zfrp8 controls cell proliferation in flies. We will use our results from Drosophila to study PDCD2 in humans. Our studies in humans will be enhanced by our studies in the fly, thus providing synergy between these two systems. Our aims are: 1. It is unclear why the lack of zfrp8 results in lymph gland overgrowth, but generally slows the growth of other tissues. In situ hybridization shows that the transcript is uniformly distributed in early embryos suggesting that the specificity comes from Zfrp8 functioning together with other lymph- gland-specific factors. The overgrowth is first seen in stage 16-17 embryos, when the number of lymph gland cells is about double that in wild type (Fig. 6, [56]). This phenotype is due either to an enlargement of the lymph gland progenitor resulting from abnormal morphogenetic signaling. Alternatively, and perhaps more likely, the precursors undergo about one additional round of division before stages 16-17 of embryogenesis. In order to distinguish between these possibilities and to elucidate zfrp8 function in early development, we propose studying the early development of the lymph gland using appropriate markers. The present model says that the lymphgland medulla contains stem cells that ultimately differentiate into three different hemocytes. Based on the zfrp8 mutant phenotype we predict that hematopoiesis is more involved and closer to the human model. We propose to investigate normal development of the lymnphgland by inducing marked clones at different stages of development. We have previously identified pannier (pnr), a GATA factor, as a suppressor of the zfrp8 phenotype. To further characterize the opposing functions of zfrp8 and pannier in lymph gland development, we propose to perform clonal analysis of mutant lymph gland cells and genetic interaction studies of zfrp8 and pannier. 2. Our preliminary studies in clinical samples and human cell lines show that PDCD2 expression in humans is more complicated than in Drosophila where there is one zfrp8/PDCD2 transcript that gives rise to one protein;there are multiple human PDCD2 cDNAs, giving rise to at least two protein isoforms, differentially expressed both in normal and malignant human tissues. The smaller 44kD PDCD2 protein, we call PDCD2[unreadable]33, is barely detected in normal blood samples but is highly expressed in human cancer cell lines and clinical blood samples isolated from patients with hematologic malignancies. The larger, 48kD isoform is not detected in these leukemic samples, but is the only PDCD2 isoform present in normal human bone marrow cells, implying that the 48kD protein plays a role in normal hematopoiesis. In support of this idea, analysis of bone marrow from a leukemia patient who achieved a complete clinical remission as a result of therapy, shows predominate expression of the 48kD protein, while the lower 44kD isoform, overexpressed in leukemic blasts, is barely detected once normal hematopind high levels of a smaller form of the protein (44kD), barely detected in blood from healthy controls. This smaller form of PDCD2 is strongly present in all cancer cell lines we haveilar function as its larger isoform, but overexpression of this form may result in a gain-of-function over-proliferative or oncogenic phenotype. To study the nature of the different PDCD2 mRNAs and protein forms we will amplify the transcripts from normal and malignant tissues by RT-PCR, determine their sequence, and investigate if additional mutations or variations are present. We will also establish the amino acid sequence of the protein forms and determine their subcellular localization. We are aiming at developing a PCR approach to differentiate between the forms of PDCD2 found in normal bone marrow and blood, and the PDCD2 forms associated with leukemic cells.